Oxidative damage to tissues by hydrogen peroxide and PUFA hydroperoxides has become a recurring theme as a mechanism for the induction of a variety of medical conditions including myocardial ischemia, cancer, and aging. In addition to lipids, proteins are a target of such damage. Myoglobin, a heme protein that is ubiquitous in aerobic muscle tissues, has peroxidase activity that can result in oxidative damage to a variety of biological molecules, including itself and membrane lipids. We are now concluding a series of investigations of myoglobin-derived radicals, which has given a rather complete understanding of the four types of radicals formed in myoglobin and their interrelationships. Because mitochondria are the primary site of hydrogen peroxide generation in vivo, we have initiated investigations of cytochrome c oxidase (CcO)-derived radical formation. CcO, the terminal enzyme of the mitochondrial respiratory chain, is a multicomponent membrane protein with a molecular weight of 200,000 comprising 13 different polypeptide subunits. The enzyme functions to reduce dioxygen to H2O at the active center of heme a3-CuB and couples proton pumping across the mitochondrial inner membrane to provide the driving energy for ATP synthesis. It has been reported that a peroxidase-like reaction can occur at the heme a3-CuB center of CcO. The addition of excess H2O2 can oxidize CcO to yield peroxy intermediate, or compound P, and one-electron reduction of compound P with either ferrocyanide or cytochrome c yields the ferryl-oxo species, or compound F. Finally, one-electron reduction of compound F returns the CcO to the oxidized state and completes the catalytic cycle. The structure of compound P remains unclear and is under debate . Compound F was proposed to be structurally related to an HRP-compound II-like species. One would expect that both compound P and compound F are highly reactive if the chemistry of the H2O2/CcO system is similar to that of peroxidases. However, no direct evidence indicates that the compound P or compound F species of CcO can oxidize the protein and initiate free radical-mediated lipid peroxidation as seen with metmyoglobin and cytochrome c. - hydrogen peroxide, myoglobin, cytochrome c oxidase